Tobacco addiction remains a leading preventable cause of premature deaths worldwide, and the long- term efficacy of current smoking cessation treatments is modest. The primary goal of the proposed project is to develop a reliable and minimally invasive bioassay of nicotine exposure that could be useful in assessing therapeutic-induced changes in nicotine intake and evaluating subjects' compliance with treatment regimens in clinical trials of smoking cessation therapies and new tobacco products with reduced nicotine content. Currently the best technique for quantification of nicotine exposure is assessment of plasma cotinine. However, the half-life of cotinine is ca. 16-h. Therefore, to provide reliable dat on cumulative nicotine exposure over several days, cotinine assays should be performed daily; otherwise a biomarker with a longer half-life is needed. A potentially useful long-lived biomarker for nicotine exposure is the nicotinic receptor (nAChR) upregulation in blood granulocytes (half-life ca. 5 days). A small but robust body of studies performed with [3H]-nicotine and [3H]-epibatidine has provided support for this promising measure. A serious drawback of these approaches is a requirement for large blood volume (ca. 40 mL). To minimize the amount of blood needed, we propose to use nAChR radioligands labeled with Iodine-125. Because the specific radioactivities of [125I]-radioligands are ca. 20 times those of [3H]-radioligands, we predict that the use of [125I]-labeled radioligands would allow us to use 1/20 of the blood volume needed for the assays with tritiated ligands. We also hypothesize that lymphocytes might be more useful for assessment of cumulative nicotine exposure because they have a much longer lifespan and likely higher expression of ?2-containing (?2*) nAChRs than granulocytes. Finally we hypothesize that the level of upregulated ?4* nAChR subtypes in smoker granulocytes is even greater than that for ?2* subtypes. To address these hypotheses we propose the following Specific Aims: Aim 1: To determine the minimum blood volume required for quantification of ?2* and ?4* nAChRs in human granulocytes and lymphocytes using 5-[125I]I-A-85380 and 125I-labeled epibatidine (125I-EB). Aim 2: To assess upregulation of ?4* and ?2* nAChRs in smokers' granulocytes and lymphocytes; Aim 3: To assess the relationship between upregulation of ?2* and ?4* nAChRs in white blood cells and nicotine exposure, assessed by nicotine intake per day and plasma cotinine level. To our knowledge, this project would be the first study to characterize white blood cell nAChRs using 5-[125I]I-A-85380 and 125I-EB and would be the first one to determine the extent of ?4* nAChR upregulation in smokers' white blood cells. The project is also technologically innovative in the utilization of several novel approaches. Successful completion of this study would provide critical data for the future assessment of nAChR upregulation in white blood cells as a valuable biomarker of extended nicotine exposure.